Lactococcus lactis’ Effect on the Intestinal Microbiota of Streptococcus agalactiae-Infected Zebrafish (Danio rerio)

ABSTRACT Streptococcus agalactiae is a common pathogen in aquaculture that disrupts the balance of the intestinal microbiota and threatens fish health, causing enormous losses to the aquaculture industry. In this study, we isolated and screened a Lactococcus lactis KUST48 (LLK48) strain with antibacterial effect against S. agalactiae in vitro and used it as a potential probiotic to explore its therapeutic effect on zebrafish (Danio rerio) infected with S. agalactiae. This study divided zebrafish into 3 groups: control group, injected with phosphate-buffered saline; infection group, injected with S. agalactiae; and treatment group, treated with LLK48 after S. agalactiae injection. Then, the 16S rRNA gene sequences of the intestinal microbiota of these 3 groups were sequenced using Illumina high-throughput sequencing technology. The results showed that the relative abundance of intestinal bacteria was significantly decreased in the infection group, and a high relative abundance of S. agalactiae was observed. The relative abundance of the intestinal microbiota was increased in the treatment group, with a decrease in the relative abundance of S. agalactiae compared to that in the control group. In the Cluster of Orthologous Groups of proteins function classification, the relative abundance of each biological function in the infection group was significantly lower than that of the control and treatment groups, showing that LLK48 has a positive therapeutic effect on zebrafish infected with S. agalactiae. This study provides a foundation for exploring the pathogenic mechanism of S. agalactiae on fish and their intestinal symbionts, and also presents a new approach for the treatment of S. agalactiae infections in fish aquaculture systems. IMPORTANCE L. lactis KUST48 (LLK48) with a bacteriostatic effect against S. agalactiae was isolated from tilapia intestinal tracts. S. agalactiae infection significantly reduced the relative abundance of intestinal bacteria and various physiological functions in zebrafish intestines. LLK48 demonstrated infection and subsequent therapeutic effects on the S. agalactiae infection in the zebrafish intestine. Therefore, the potential probiotic LLK48 can be considered as a therapeutic treatment for S. agalactiae infections in aquaculture, which can reduce the use of antibiotics and help maintain fish health.


Introduction
• The Introduction is very poorly written and needs to be rewritten. Some examples: lines 46-49; 77-78; 81-87; 88-91; etc. • Line 60, the bibliographic citation (6) is not adequate. Please use a proper citation. • Lines 64-66. Bibliographic citation (9) does not support the written sentence. • Lines 60-63. The bibliographic citation is not adequate. There are several papers about of the beneficial uses of probiotics in Aquaculture. You must use an appropriate citation. • Lines 74-76: this sentence is confusing; please indicate if these data are being published in another journal or are part of this work.
• Lines 108-118. This information should be in materials and methods section.
Materials and methods: Section 2.1 • Please change the title: "Isolation of intestinal flora" by "LAB isolation from tilapia intestinal tract". • Please add the proportion of the dilutions performed for the isolation assay.
Section 2.2: in this section there are several mistakes. Please check it carefully • I suggest changing the title: "Screening of antibacterial strains" by "Screening of strains with antimicrobial activity". • Please add the origin and culture conditions of the S. agalactiae strain. • Please add the dilutions performed for the minimum inhibitory concentration (MIC) assay. • Line 141: please clarify if it is broth or semi-solid medium, if it is semi-solid medium, indicate the agar concentration. • Lines 142: does the plate really invert to mix? • Line 145: Please add Kanamycin concentration. Section 2.3: • Line 152-153: Please review the content of the following sentence: "In order to select a lactic acid bacterial strain with good antimicrobial activity against S. agalactiae, total DNA was extracted using a DNA Extraction Kit..." • Line 155: "...of 16S gene sequencing...", please replace with "...of 16S rRNA gene sequencing..." • Line: 165-167: Please review the following sentence: "The sequenced 16S was searched in GenBank of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/)." Section 2.4: • I suggest changing the title for "Zebrafish and experimental design" • Lines 178-181: The content of these lines should be in section 2.2. • Line 192: "Thirty zebrafish in each group were then randomly divided into three special aquarium divider box...". Please explain which groups you are referring to. Section 2.5 • In lines 210-212 the text is confusing: "...the intestines of 15 zebrafish were randomly divided into 5 portions, and each portion of intestine was mixed into one sequencing sample for each group". Please rewrite this sentence and explain better how each sample was prepared.
• In materials and methods, please indicate the period of time used to calculate the mortality rate In materials and methods, please add a new section that details the prediction method of the functional changes of the intestinal microbial community (PICRUSt). Section 2.8 • Please add the statistical analysis of the predicted functions using PICRUSt.

RESULTS
Section 3.1: -Please change the title: "Isolation and bacteriostatic experiment of intestinal bacteria" by "Isolation and antimicrobial activity of intestinal bacteria". -Throughout the manuscript, in the objective of this study (lines 103-104), in section 2.1 of materials and methods and in the title of section 3.1 of results, several isolated bacteria are mentioned (several isolates belonging to the group of LAB), however the result of only one is showed. I suggest adding a table with the results of antimicrobial activity against S. agalacteae of the 79 LAB strains isolated from tilapia intestine. Please add also the results of the minimum inhibitory concentration (MIC) described in section 2.2 of materials and methods. -In figure legends, for Figure 1, it would be appropriate to change the name of the strain "LLK48" to "strain KUST48" since at this point the strain has not yet been identified. -Line 269: there are several mistakes in grammar and sentence construction. Please check it carefully. Moreover, please change "...the 16S rDNA sequence..." by "...16S rRNA sequence..." Section 3.2: -In this section (lines 276-287) there are several mistakes in grammar and sentence construction. Please check it carefully. Section 3.3 -Please change the title: "Optimization analysis of high-throughput sequencing data" by "Analysis of sequences". Section 3.4 -Lines 306-307. Please, check "Actinobacteria and Fusobacteria" are repeated. -There are several grammatical mistakes in this paragraph. Please check it carefully Section 3.5 -Lines 316. "Escherichia-Shigella and Aeromonas were the main genera (the sum of the relative abundance > 0.1%) in all the three groups ( Section 3.7 -Lines 369-370. "The analysis results of species relative abundance in each group at the genus level are shown in Figure 5". Please check this sentence, in Figure 5 the analysis of microbial community composition at the phylum level is showed.
-The results in the section 3.7 are similar to that shown in the section 3.5, please unify the results in a single section.
-The results are very poorly presented. The manuscript has many grammatical mistakes, making it very difficult to read. -It would be interesting to analyze the relative abundance of specific bacterial genera such as Vibrio sp., Lactococcus sp., for the different groups Section 3.8 -Please indicate the significant differences in predicted microbial functions among the three groups. This section was poorly written. Please rewrite it. Table 2: -"Different superscripts identify significant differences (P < 0.05) in relative abundance". Please indicate whether the significant differences are within the same group or between groups.  References not formatted correctly. Species names not formatted correctly. Some article titles in caps, others in lowercase (be consistent). Lines 507, 519, 558 spacing errors. 549 incomplete citation. I can't read Table 1 lines 619, 624 spacing errors figure 6 very few of these phyla are visible here Reviewer #3 (Comments for the Author): The study is generally appropriate, but the authors need to have appropriate answers to the following questions: -The stages of laboratory evaluations in the materials and methods section do not match the results section. Lactobacilli should be screened first and then the antimicrobial activity should be checked. LLK48 is introduced before the screening.
-In a separate group, uninfected fish could be evaluated with probiotic bacteria as controls.
-In a separate group, a pretreatment with probiotic bacteria could be evaluated for its influence on the onset and severity of the infection.
-On what basis was the number of probiotic inoculated bacteria selected? -Why have growth performance and immunomodulatory function not been evaluated?
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Overall Recommendation:
The authors isolated a strain of Lactococcus lactis KUST48 from tilapia with in vitro antimicrobial activity against Streptococcus agalactiae. They studied the effect of L. lactis strain on zebrafish infected with S. agalactiae from the perspective of the intestinal microbiota. The in vivo experimental test was divided into three groups: the control group (A) injected with PBS, the infected group (B) injected with S. agalactiae, and the treated group (C) injected with S. agalactiae and 24h post-injection with L. lactis KUST48. Then, the 16S rRNA gene sequences of the intestinal microbiota of these three groups were sequenced using Illumina high-throughput sequencing technology.
The topic of this study is very interesting. The results of this study showed that the injection of L. lactis KUST48 produced benefits on zebrafish infected with S. agalactiae. However, the authors refer to L. lactis as a probiotic when this was not yet demonstrated (see Abstract review). Moreover, the term "treatment" referred in the Title would not be appropriate since a more comprehensive evaluation is needed, where different doses, times, and effects of L. lactis on infected zebrafish are tested or evaluated. On the other hand, the manuscript was hard to read, the authors should find a native speaker to polish the English.
For a better understanding of the effect of L. lactis KUST48 on the zebrafish intestinal microbiota, another control group with the L. lactis KUST48 and without the S. agalactiae would be useful to add.

Title:
-I suggest the following title: Lactococcus lactis effect on the intestinal microbiota of Streptococcus agalactiae infected zebrafish

Abstract:
-The abstract is very poorly written and needs to be rewritten.
-Lines 39-41. In importance section "…The probiotic Lactococcus lactis (LLK48) can be considered as a therapeutic and water quality modifying agent for Streptococcus agalactiae (SA) infections …".
• Line 60, the bibliographic citation (6) is not adequate. Please use a proper citation.
• Lines 60-63. The bibliographic citation is not adequate. There are several papers about of the beneficial uses of probiotics in Aquaculture. You must use an appropriate citation.
• Lines 74-76: this sentence is confusing; please indicate if these data are being published in another journal or are part of this work.
• Lines 108-118. This information should be in materials and methods section.

Materials and methods:
Section 2.1 • Please change the title: "Isolation of intestinal flora" by "LAB isolation from tilapia intestinal tract". • Please add the proportion of the dilutions performed for the isolation assay. • Please add the dilutions performed for the minimum inhibitory concentration (MIC) assay.
• Line 141: please clarify if it is broth or semi-solid medium, if it is semi-solid medium, indicate the agar concentration.
• Lines 142: does the plate really invert to mix?
Section 2.3: • Line 152-153: Please review the content of the following sentence: "In order to select a lactic acid bacterial strain with good antimicrobial activity against S. agalactiae, total DNA was extracted using a DNA Extraction Kit…" • Line 155: "…of 16S gene sequencing…", please replace with "…of 16S rRNA gene sequencing…" • Line: 165-167: Please review the following sentence: "The sequenced 16S was searched in GenBank of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/)." Section 2.4: • I suggest changing the title for "Zebrafish and experimental design" • Lines 178-181: The content of these lines should be in section 2.2.
• Line 192: "Thirty zebrafish in each group were then randomly divided into three special aquarium divider box…". Please explain which groups you are referring to.
Section 2.5 • In lines 210-212 the text is confusing: "…the intestines of 15 zebrafish were randomly divided into 5 portions, and each portion of intestine was mixed into one sequencing sample for each group". Please rewrite this sentence and explain better how each sample was prepared.
• In materials and methods, please indicate the period of time used to calculate the mortality rate In materials and methods, please add a new section that details the prediction method of the functional changes of the intestinal microbial community (PICRUSt).
Section 2.8 • Please add the statistical analysis of the predicted functions using PICRUSt.

Results
Section 3.1: -Please change the title: "Isolation and bacteriostatic experiment of intestinal bacteria" by "Isolation and antimicrobial activity of intestinal bacteria".
-Throughout the manuscript, in the objective of this study (lines 103-104), in section 2.1 of materials and methods and in the title of section 3.1 of results, several isolated bacteria are mentioned (several isolates belonging to the group of LAB), however the result of only one is showed. I suggest adding a table with the results of antimicrobial activity against S. agalacteae of the 79 LAB strains isolated from tilapia intestine. Please add also the results of the minimum inhibitory concentration (MIC) described in section 2.2 of materials and methods.
-In figure legends, for Figure 1, it would be appropriate to change the name of the strain "LLK48" to "strain KUST48" since at this point the strain has not yet been identified.
-Line 269: there are several mistakes in grammar and sentence construction. Please check it carefully. Moreover, please change "…the 16S rDNA sequence…" by "…16S rRNA sequence…"  Section 3.7 -Lines 369-370. "The analysis results of species relative abundance in each group at the genus level are shown in Figure 5". Please check this sentence, in Figure 5 the analysis of microbial community composition at the phylum level is showed. -The results in the section 3.7 are similar to that shown in the section 3.5, please unify the results in a single section. -The results are very poorly presented. The manuscript has many grammatical mistakes, making it very difficult to read. -It would be interesting to analyze the relative abundance of specific bacterial genera such as Vibrio sp., Lactococcus sp., for the different groups Section 3.8 -Please indicate the significant differences in predicted microbial functions among the three groups. This section was poorly written. Please rewrite it. -"Different superscripts identify significant differences (P < 0.05) in relative abundance". Please indicate whether the significant differences are within the same group or between groups. -Please indicate the meaning of the categories (A, B, C, etc.) and the values that appear in Table 3. Indicate the results of the statistical analysis among 3 groups.  4.The abstract is very poorly written and needs to be rewritten.

Response:
We have revised the ABSTRACT, as suggested.
5.Lines 39-41. In importance section "...The probiotic Lactococcus lactis (LLK48) can be consideredas a therapeutic and water quality modifying agent for Streptococcus agalactiae (SA) infections ...".-It is not appropriate to speak of "probiotic" (throughout the manuscript), because both, other testsare needed to confirm that a bacterial strain can be considered a probiotic (i.e. antibiotic resistancegenes, haemolytic activity, bile tolerance, etc.), and the intraperitoneal injection is not the properadministration way. A probiotic is administered with the food or in the rearing water. Thus, theyshould change "probiotic" by "potential probiotic".

Response:
Thank for the reviewer's reminder. We have changed probiotics to potential probiotics. Please see lines 38.
6.In the present study, the effect of L. lactis (LLK48) on water quality was not demonstrated norevaluated. Please check this. 9.Line 60, the bibliographic citation (6) is not adequate. Please use a proper citation.

Response
Response:According to the reviewers' opinions, references have been supplemented to explain. Please see lines 58.
10.Lines 60-63. The bibliographic citation is not adequate. There are several papers about of the beneficial uses of probiotics in Aquaculture. You must use an appropriate citation.